Electrophoretic in situ tissue staining

ABSTRACT

The present invention introduces a radically different way of accelerating biomolecule conjugates into tissue, and hence towards their targets for purposes of tissue staining. The invention provides for an order of magnitude improvement over the prior art diffusion process used to stain tissue. The invention comprises a method of tissue staining by applying an electric field to a tissue sample in the presence of an electrolyte and biomolecular conjugates of interest suspended in the electrolyte. Typical staining times are reduced to seconds as opposed to 30-120 minutes common in the prior art. The invention is also directed to devices for performing the method.

BACKGROUND

1. Field of the Invention

This invention relates generally to the field of automated tissuestaining apparatus, and in particular is a new method of introducingstains into tissue using electrophoresis.

2. Description of Related Art

Tissue staining is an ancient art by modem standards that goes back overone hundred years. Recently, efforts have been made to automate theprocedure of applying different types of chemical and biochemical stainsto tissue sections. Instruments that have been invented for this purposeinclude the Ventana Medical Systems' line of dual carousel-basedinstruments such as the 320, ES®, NexES®, BENCHMARK®, and the BENCHMARK®XT. Patents that describe these systems include U.S. Pat. Nos.5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which areincorporated herein by reference in their entirety. Another type ofautomated stainer is the TechMate® line of stainers, described in U.S.Pat. Nos. 5,355,439 and 5,737,499, both of which are incorporated hereinby reference in their entireties.

The rate of Immunohistochemical and in situ hybridization staining ofmicrotome-sectioned tissue on a glass slide is limited by the speed atwhich the biomolecules of interest can diffuse into the tissue from anaqueous solution placed in contact with the tissue section. Intacttissue presents many barriers to diffusion such as the lipid bilayermembranes that enclose individual cells and organelles, and the effectsof cross-linking that the fixation process generates. The proteinantibody or DNA probe molecules of interest are relatively large,ranging in size from a few kilo Daltons to several hundred kilo Daltons,which causes them to diffuse slowly into solid tissue with typical timesfor sufficient diffusion being in the range of several minutes to a fewhours. A typical incubation period is thirty minutes at 37 degreescentigrade.

The diffusion rate is driven by concentration gradient so the rate canbe increased by increasing the concentration of the conjugate in thereagent. However, this has two detrimental effects. First, theconjugates are often very expensive, so increasing their concentrationis wasteful and not economically viable. Second, the excessive amount ofconjugate that is driven into the tissue, when high concentrations areused, gets trapped in the tissue, and cannot be rinsed out and causeshigh levels of background staining. This background staining is callednon-specific staining and, in an informational sense, is just noise. Inorder to reduce the noise and increase the signal of specific staining,low concentrations of conjugate are used with long incubation times toallow the conjugate to find and bind to only the specific sites.

Electrophoresis is an electrochemical separation technology commonlyapplied to separate biological molecules on the basis of theircharge-to-mass ratio. Generally, a gel slab is prepared from a suitablepolymeric material such as polyacrylamide by adding water to it insufficient amount to create a semi-solid gelatinous slab. This is thematrix used to both contain the sample to be separated, and transmit theelectric current used to electromotively move the various chargedmolecules. The pH of the gel can be manipulated to charge a biomoleculethat is otherwise uncharged, thereby giving it the prerequisite netcharge so that it will move when a field is applied to it. When the gelhas an electric field applied to it, the charged molecules will migratethrough the gel towards their opposite pole, i.e., negatively chargedbiomolecules will move towards the positive pole, and vice versa. Theprocess is very commonly used in the biological research field toseparate complex mixtures, and is termed “PAGE” (Polyacrylamide gelelectrophoresis). A related technology is capillary electrophoresis(“CE”), which is the same basic electrochemical separation performed inthin glass capillary lumens filled with an electrolytic solution.

There continues to be a need for faster introduction of biomoleculesinto tissue sections, and for lower amounts of non-specific backgroundstaining.

SUMMARY OF THE INVENTION

The present invention introduces a radically different way ofaccelerating biomolecule conjugates into tissue for purposes of tissuestaining, and hence towards their targets. The invention provides for anorder of magnitude improvement over the prior art diffusion process usedto stain tissue. The invention comprises a method of tissue staining byapplying an electric field to a tissue sample in the presence of anelectrolyte and a biomolecular conjugate molecule of interest suspendedin the electrolyte. Typical staining times are reduced to seconds asopposed to 30-120 minutes common in the prior art.

It is an object of this invention to accelerate the movement ofconjugate molecules from the aqueous solution into the solid tissue.Another object is to reduce the background staining due to conjugatesthat are not bound to specific sites. A further object is to reduce theconcentration of the conjugate required in the reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-sectional view of an apparatus using this method.It uses electrophoresis to cause molecules to pass into and through athin cut piece of tissue.

FIG. 2 shows an ITO coated slide with a capillary gap.

FIG. 3 is an ITO coated slide with a moving upper electrode shown overthe slide.

FIG. 4 is a cross-section through the movable upper electrode.

FIG. 5 is another cross-section through the movable dual electrode ofembodiment four having incorporated conductive rods.

FIG. 6 is a schematic of wells and tissue positions in an agarose gel.

FIG. 7 is a photomicrograph of Tissue Section 1.

FIG. 8 is a photomicrograph of Tissue Section 2.

FIG. 9 is a photomicrograph of Tissue Section 3.

FIG. 10 is a photomicrograph of Tissue Section 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is directed to a method of introducing a conjugatemolecule into tissue comprising applying an electric field to the tissuein the presence of an electrolyte and a conjugate molecule of interestsuspended in the electrolyte. A conjugate molecule may be any moleculethat has a complementary binding portion that, when brought intoproximity to its complementary binding site, binds to the site.Antibodies having complementarity determining regions, and DNA oligomersthat have matching sequences to their target DNA, are two examples ofconjugate molecules. The conjugate molecules of interest are all chargedwhen dissolved in an aqueous solution of electrolyte of the correct pH.The net charge facilitates their movement through the electrolytesolution by the electric field. Tissue includes both tissue sections andintact cells prepared according to conventional methods such ascytospins or Thin Preps.

The technology generally known as Electrophoresis has been used for manyyears, both in research and industry to separate molecules of differingsizes and charges. Descriptions for the use of electrophoresis are givenin U.S. Pat. Nos. 2,992,979; 3,384,564; 3,494,846; 3,677,930; 3,844,926;5,382,522 and 5,536,382 among others. The prior art describes applyingthe electric field across a liquid or gelatinous material, such asagrose, while the solution containing the molecules of interest isplaced at one end. The molecules of interest migrate through thematerial, at rates that depend on their net charge and molecularweights. Some of the prior art discloses the use of electrophoresis toseparate human biomolecules for clinical applications. In U.S. Pat. No.5,536,382, methods are provided for the analysis of constituents ofhuman biological fluids using capillary electrophoresis. A clinicalsample was mixed with a labeled reagent which specifically binds theanalyte of interest. Capillary electrophoresis is then used to resolvebound from unbound reagent, and the constituents quantitated bymeasuring directly or indirectly the amount of bound reagent. In U.S.Pat. No. 5,382,522, a serum or plasma sample was assayed to determinethe concentration of two different analytes selected from the groupconsisting of creatine kinase-MB species and creatine kinase-BB species.However, none of the prior art uses an electric field to move moleculesinto human tissue.

The most general description of this invention is that it is any methodthat applies an electric field across both an aqueous solutioncontaining conjugate molecules and some tissue of interest in order touse the electrophoretic forces to drive the conjugate molecules into thetissue. In the preferred embodiment, the tissue is human tissue that issuspected of harboring some disease and has been cut on a microtome to athin section. However, cell preparations comprising intact cells adheredto a flat surface for further processing are also encompassed by thisgeneral method. A thin section is generally between two and thirtymicrons thick. There are several different ways to apply the electricfield to thin cut tissue, three of which are described below.

A first preferred method is to mount the thin cut tissue on a porousmembrane, apply a conductive aqueous fluid to both sides, add reagentcontaining the conjugate into the fluid on at least one side, placeelectrodes on opposite sides and apply an electric field between theelectrodes. Direct current is the preferred mode of generating theelectric field, but alternating current may also be used. FIG. 1 shows across sectional view of an apparatus using this method. It useselectrophoresis to cause molecules to pass into and through a thin cutpiece of tissue.

The tissue 11 is attached to a porous membrane 3. The tissue can be fromany area of the body, but tests have been run using tonsil. The membranecan be made from any hydrophilic, porous material. One method that hasbeen tried is to use PTFE film, commonly called “plumber's tape”. ThePTFE film must me made hydrophilic by polymerizing polyvinyl alcohol toits surface before the tissue will bond to it. The lower electrode 5 ismade from a solid disk of metal, preferably 316 SS and is placed intothe bottom of the five millimeter deep depression in the lower ring, 1.This depression forms a basin below the membrane 3. An electrical lead,not shown, is attached to the lower electrode and passes out through thelower ring through a sealed hole, not shown, and is connected to one legof the electrophoresis power supply, not shown. The membrane isstretched over the top of the lower ring, and down over its outer,tapered diameter. The membrane is retained by pressing the intermediatering 8 over the lower ring 1 trapping the membrane 3 between the twotapered diametrical surfaces. The upper ring 2 is pressed onto theintermediate ring 8 forming another five millimeter deep basin, this onebeing above the membrane 3. This upper basin is hydraulically connectedto the lower basin by means of two fittings 9 and a section of tubing 7.The fittings 9 are standard barb fittings made of thermoplastic and thetubing 7 is standard Tygon. The upper electrode, 6, is made of stainlesssteel wire mesh which allows reagent to be poured into the upper basinand keeps the top surface of membrane, 3, and the tissue, 11, visible.Upper electrode, 6, is connected to the electrophoresis power supply,not shown, by means of wire, 4. Another section of Tygon tubing, 10, isconnected to a third barbed fitting, 9, which bleeds air out of thelower basin as fluid is poured into the upper basin. In operation, theupper basin is filled with conductive reagent, such as Tris-Acetate EDTAbuffer at 10% concentration. This reagent also flows into the lowerbasin, displacing the air through the passages leading to tubing, 10.After the basins are filled, a conjugate is placed into the upper basin.Tests have been run using anti-CD34 antibody which attaches to capillarytissue in the tonsil tissue. The anti-CD34 is first mixed 1:1 withglycerol so that is sinks through the Tris buffer to the top of thetissue and the membrane. An electric potential of ten volts is appliedacross the ten millimeters of distance between the electrodes, providingan electric field with a strength of 100 volts per meter. The anti-CD34antibody moves through the five micron thick tissue in less than tenseconds. The apparatus is disassembled, and the area of the tissue iscut out of the membrane. It is then processed with a standard chromagindetection kit. The capillaries in the tissue stand out against thebackground.

If a membrane is used to support the tissue during electrophoresis, themembrane containing the tissue must be removed from its supportstructure, applied to a glass slide and coverslipped. In the preferredembodiment, the membrane must be transparent after it is coverslipped.In order for the membrane to be transparent after coverslipping, it musthave an index of refraction that is very near that of the coverslipmedia. Standard, xylene soluble coverslip media, such as Super-Mount™,has an index of refraction of 1.54 which is very close to that oftypical proteins in human tissue. Membranes that have an index ofrefraction close to this are PET and nylon 6.

A second preferred method is to apply an electric field across theaqueous solution and the thin cut tissue of interest is to coat theglass slide with a conductive layer, apply the tissue directly to thetop of the conductive layer, add a conductive reagent of the correct pHthat contains the conjugate molecules of interest over the top of thetissue, cover the conductive reagent with a second electrode and thenapply a potential between the conductive layer on the slide and theupper conductive electrode. After the conjugate has been driven into thetissue and sufficient time has elapsed for the conjugates to find theirspecific sites (a few seconds at most), the electric potential can bereversed, so that any unbound conjugates are driven out, reducing thebackground noise of non-specific binding.

The conductive layer needs to be transparent so that after the stainingis complete, a pathologist can look at the tissue through a microscopewith the tissue illuminated from below. Two possible candidates for aconductive, transparent film are gold and ITO (Indium Tin Oxide). Bothare applied as very thin layers in a vacuum chamber. Any material thatis both transparent, conductive and resistant to oxidation can be used.

FIG. 2 shows an apparatus for applying an electrical field across acapillary gap of reagent that contains conjugate molecules and across athin cut layer of tissue that is adhered to an ITO coated glass slide22. All the components are attached to a non-conductive base plate, 21,made from Ultem® 1000. The microscope slide, 22, is retained in thefixed clamping fixture, 23, by the force exerted by thumb screw, 24. Allof the clamping fixture, 23, is made of conductive material, such asstainless steel. The tissue, 25, is adhered to the top of the ITOsurface of slide 22. The upper electrode, 26, is clamped into slidingclamping fixture, 27, which is also made of stainless steel and slidesin a groove in backing plate 28. The size of the capillary gap betweenthe slide 22 and the upper electrode 26 is adjusted by screw 29 which isthreaded into sliding clamp 27 and pushes against the top surface ofbase 21. The wire leads, 30, 31 are connected to the electrophoresispower supply (not shown).

The resistance of an ITO coated surface is about 15 ohms per squareinch. The slides are 25 mm wide and have 50 mm of length extending fromthe fixed clamp, 2. This means that the resistance of the film along thelength of the 50 mm of extended slide is 30 ohms. The resistance of thecapillary gap is much less, being about 0.33 ohm for a 200 μm thick gapof reagent. In order for the electric field across the gap to beconstant, the linear resistance of the upper electrode must match thatof the ITO coating. This can be done by using another ITO coated slideas the top electrode or by using a platinum or gold coated slide thathas the same resistance as the slide coating. The potential that needsto be applied depends on the resistance of the coatings and fluid, thelength of overlap and the resistance of the capillary gap. Theelectrical potential is applied to the capillary gap by connecting thewires to a power supply. In order to produce a uniform electric field ofone volt per millimeter over a 200 μm gap (0.20 volt), a potential of 24volts is required across the electrodes.

A third preferred method of applying the required potential across thereagent and tissue is to use a curved, movable upper electrode, as shownin FIGS. 3 and 4 in conjunction with an ITO coated microscope slide 22.The slide 22 is clamped in the fixed clamp 23 as in the previousembodiment. However, instead of a fixed upper electrode 26 the movingupper electrode 40, is attached to an air cylinder 45 that moves itlengthwise along the slide. The moving upper electrode 40 is 25 mm wideand has a curved lower surface that is stepped. The outer rims 41 of themovable electrode 40 are one millimeter wide at both sides and extendradially 200 μm beyond the curved lower surface 42 (see FIG. 4) whichlies between the two rims 41.

The two rims 41 slide on the surface of the slide while the raisedsurface 42 is approximately 200 μm above the slide. The movableelectrode 40 is made of a non-conductor such as Ultem® 1000. Its curvedlower surface 42 lies between the rims 41 and is plated with platinumand is electrically connected to the lead wire 43 which in turn issecured to the Ultem electrode 40 by means of screw 44. Tissue 25 isadhered to the ITO surface of slide 22 and a small volume of about 15 μlof the reagent that contains the conjugates of interest is placed on theslide from a pipette (not shown). The air cylinder 45 pushes the movableelectrode 40 onto the slide where it contacts the 15 μl puddle ofreagent. The reagent is attracted to the lower platinum-plated surface42 of the moveable electrode 40 forming a meniscus 46. The surfacetension of the reagent strongly attracts the reagent to theplatinum-plated surface 42 and the top of the slide 22, and retains itthere while the electrode 40 is moved axially along the slide 22 by theair cylinder 45. The reagent wets the top surface of the slide and thetissue as it slides across them and the electric potential provides theelectrophoretic force that drives the molecules into the tissue. Thereagent is strongly mixed by the shear forces in the reagent as theelectrode moves. With this apparatus, the potential can be reversed todrive out conjugate that is not bound to specific sites.

Even though the resistance of the ITO on the slide between the electrodeand the clamped end of the slide varies significantly, a constantpotential is maintained between the platinum coated surface and the ITOsurface of the slide by means of a constant current circuit thatsupplies power to the two wires. A constant current circuit is awell-known device to those skilled in the art of transistor circuitry.

The reagents used in any step need to be removed before reagents for thenext step are applied. This is accomplished in this embodiment bybringing the movable electrode 40 off of the slide 22 and onto rinseblock 47. Rinse block 47 has holes in its upper surface that are fed bytubing 48. Rinse fluid to the rinse block 47 is controlled by a valve,not shown. Electrode 40 is rinsed at the rinse block 47 then, while itis covered with rinse solution, it is returned to the slide 22. On theslide it picks up more reagent, and is again returned to the rinse block47. By a series of these motions, the reagent on the slide is seriallydiluted until it is sufficiently dilute as not to cause any interferencewith the next reagent.

A fourth preferred method (shown in FIG. 5) of applying a potentialacross the tissue is similar to method three but does not use aconductive coating on the slide. Instead of a conductive lower surfaceon the insulated movable block, two conductive rods, 51, 52, were used.The rods are located on opposite ends of the movable block 50 with theiraxes running across the narrow width of the slide. The voltage isapplied between the two rods, one rod connected to the positivepotential lead, 53 and the other connected to the negative potentiallead 54. As current flows from one rod to the other through the reagenton the slide 55, the charged molecules are driven into the tissue. As inmethod three, the block was moved up and down the length of the slidewhile the current was being applied. Rinsing of the slide may beaccomplished in the same manner as described above for method three.

Experiment 1. Electrophoretic Tissue Staining using Anti-CD34 Antibodyin Tonsil.

The following experiment was run to determine if antibody could beintroduced electrophoretically into tissue. The tissue was adhered to ahydrophilic polytetrafluoroethylene (PTFE) membrane (TEFLON® Plumber'sTape) to enable manipulation and orientation of the tissue in the gel,and then embedded in an agarose gel for subsequent electrophoresis.

Procedure: four sections of 5 μm-thick human tonsil were mounted toPVA-treated hydrophilic PTFE membrane, air dried for 48 hours, overnightdried at 60° C., manually de-paraffinized and re-hydrated (standardprocess of dipping sections sequentially in xylene, then 100% EtOH, 90%EtOH, 80% EtOH, 70% EtOH, and finally 100% H2O). The PTFE membrane wasmade hydrophilic by wetting in Isopropyl alcohol first, then soaking forseveral hours in a solution of 0.1% polyvinyl alcohol in phosphatebuffer, pH 2.2 and 5% glutaraldehyde, and rinsed in DI water. Anyhydrophilic membrane that will pass antibodies will work, however.

With regard to FIG. 6, ten wells are shown, numbered 1-10. Three of thetissue/membrane sections, shown as Tissues 2-4, were mounted in 1%agarose (GibcoBRL, Cat. No. 15510-019 in 1× TAE buffer, Sigma Cat. No.T9650) and cut out. Tissue 1 was not mounted in agarose prior to pouringthe gel and was positioned in the electrophoresis apparatus (Owl ModelB1A, flatbed) adjacent to wells 2 and 3. Tissues 2-4 were first embeddedin agarose than positioned vertically as shown in FIG. 6. The verticalpositioning places the tissue sections in the direct path of theantibodies from the wells so that the antibodies must migrate throughthe tissue under the urging of the electric field and in the directionof the large arrow at the left of FIG. 6. The apparatus was filled with1% agarose and allowed to solidify. 25 μl of anti-CD34 antibody (VentanaMedical Systems, Tucson, Ariz., Cat. No. 790-2927) was diluted 50% withglycerol (Sigma Cat No. G6279) and bromo phynol blue (Sigma Cat. No.B3269) and was added to wells 2, 3, 5, 6, 8 and 9. The electrophoresisapparatus was run at 45V for 90 minutes. An additional 25 μl ofanti-CD34 was added to wells 2 and 9 to see if additional antibody leadto increased staining, and 25 μl of FITC-labeled human IgG was added towells 1 and 10 to insure that under these test conditions the antibodywas migrating in the proper direction. The apparatus was run for anadditional 120 minutes at 45V. The tissues on the membranes were removedfrom the agarose by peeling the agarose away and a streptavidin/DABdetection kit applied manually (Ventana Medical Systems, Tucson, Ariz.,Cat. No. 760-124).

Results: Photomicrographs of the stained tissue sections correspondingto antibody from wells 2-3, 5-6, and 8-9 are shown in FIGS. 7-10. FIG. 7shows Tissue Section 1, which was in front of wells 2-3. FIG. 8 showsTissue Section 2, which was directly in front of wells 5-6. FIG. 9 showsTissue Section 3, which was in front of Tissue Section 2. FIG. 10 showsTissue Section 4, which was in front of wells 8-9. Tissue sections 1(FIG. 7) and 4 (FIG. 10) were stained equally and darker than Sections 2and 3. Section 3 was stained significantly lighter than section 2.

Conclusions:

1. Electrophoresis is able to drive anti-CD34 antibody into tonsiltissue and through tonsil tissue that is mounted on PTFE membrane.

2. The antibody binds to its antigen under these conditions.

3. The more antibody that is passed through the tissue, the darker thestain.

4. Background coloration is acceptable.

Although certain presently preferred embodiments of the invention havebeen described herein, it will be apparent to those skilled in the artto which the invention pertains that variations and modifications of thedescribed embodiments may be made without departing from the spirit andscope of the invention. For instance, although direct current isnormally used for electrophoresis, it is contemplated that alternatingcurrent could be used also. Accordingly, it is intended that theinvention be limited only to the extent required by the appended claimsand the applicable rules of law.

1. A method of introducing a conjugate molecule into tissue comprisingapplying an electric field to the tissue in the presence of anelectrolyte and a conjugate molecule of interest suspended in theelectrolyte.
 2. The method of claim 1 wherein said conjugate molecule isany molecule that has a complementary binding portion.
 3. The method ofclaim 1 wherein said conjugate molecule is selected from the groupconsisting of antibodies and polynucleotide molecules.
 4. The method ofclaim 1 wherein said tissue comprises a solid tissue sample.
 5. Themethod of claim 1 wherein said tissue comprises a cellular preparation.6. The method of claim 1 wherein the step of applying the electric fieldto the tissue comprises using alternating current.
 7. The method ofclaim 1 wherein the step of applying the electric field to the tissuecomprises using direct current.
 8. The method of claim 1 wherein thestep of applying the electric field to the tissue comprises orientingthe electric field orthogonally to the plane of the tissue.
 9. Themethod of claim 3 wherein said conjugate molecule is detectably labeled.10. The method of claim 9 wherein the labels are selected from the groupconsisting of haptens, fluorophores, radiolabels and chromophores.
 11. Adevice for electrophoretically directing conjugate molecules into atissue sample comprising: (a) a first electrode having a sample surfaceadapted for positioning and holding said tissue; (b) a second electrodespaced apart from said first electrode and defining a gap between saidsample surface and said second electrode; (c) a resevoir suitable forholding an electrolyte solution disposed on both sides of the tissuesample; and (d) means for applying an electrical current across saidsample surface whereby in response to it an electric field will formsufficient to drive the conjugate molecules into said tissue.
 12. Adevice for electrophoretically directing conjugate molecules into atissue sample comprising: (a) a first electrode having a sample surfaceadapted for positioning and holding said tissue; (b) a second electrodespaced apart from said first electrode and defining a gap between saidsample surface and said second electrode, said gap capable of supportinga meniscus of electrolye fluid; and (c) means for applying an electricalcurrent across said sample surface whereby in response to it an electricfield will form sufficient to drive the conjugate molecules into saidtissue.
 13. A device for electrophoretically directing conjugatemolecules into a tissue sample comprising: (a) a movableelectrically-insulated block, said block having at least two electrodesof opposite polarity positioned on it, said block being movable tothereby direct an electric field into said tissue sample; (b) a samplesurface adapted for positioning and holding said tissue, said samplesurface being spaced apart from said block thereby defining a gapbetween said sample surface and said block, said gap capable ofsupporting a meniscus of electrolye fluid; and (c) means for applying anelectrical current across said electrodes whereby in response to it anelectric field will form sufficient to drive the conjugate moleculesinto said tissue.